Essential role of ELOVL4 protein in very long chain fatty acid synthesis and retinal function.

Abstract

Very long chain polyunsaturated fatty acid (VLC-PUFA)-containing glycerophospholipids are highly enriched in the retina; however, details regarding the specific synthesis and function of these highly unusual retinal glycerophospholipids are lacking. Elongation of very long chain fatty acids-4 (ELOVL4) has been identified as a fatty acid elongase protein involved in the synthesis of VLC-PUFAs. Mutations in ELOVL4 have also been implicated in an autosomal dominant form of Stargardt disease (STGD3), a type of juvenile macular degeneration. We have generated photoreceptor-specific conditional knock-out mice and used high performance liquid chromatography-mass spectrometry (HPLC-MS) to examine and analyze the fatty acid composition of retinal membrane glycerophosphatidylcholine and glycerophosphatidylethanolamine species. We also used immunofluorescent staining and histology coupled with electrophysiological data to assess retinal morphology and visual response. The conditional knock-out mice showed a significant decrease in retinal glycerophospholipids containing VLC-PUFAs, specifically contained in the sn-1 position of glycerophosphatidylcholine, implicating the role of Elovl4 in their synthesis. Conditional knock-out mice were also found to have abnormal accumulation of lipid droplets and lipofuscin-like granules while demonstrating photoreceptor-specific abnormalities in visual response, indicating the critical role of Elovl4 for proper rod or cone photoreceptor function. Altogether, this study demonstrates the essential role of ELOVL4 in VLC-PUFA synthesis and retinal function.

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Essential Role of ELOVL4 Protein in Very Long Chain Fatty
Acid Synthesis and Retinal Function*
Receivedforpublication,April29,2011,andinrevisedform,December22,2011 Published,JBCPapersinPress,December24,2011,DOI10.1074/jbc.M111.256073
Richard Harkewicz‡1, Hongjun Du§¶?1, Zongzhong Tong§, Hisham Alkuraya§, Matthew Bedell§, Woong Sun§,
Xiaolei Wang§**, Yuan-Hao Hsu‡, Julian Esteve-Rudd‡‡, Guy Hughes§, Zhiguang Su¶, Ming Zhang¶,
Vanda S. Lopes‡‡, Robert S. Molday§§, David S. Williams‡‡2, Edward A. Dennis‡3, and Kang Zhang§¶4
Fromthe‡DepartmentofChemistryandBiochemistry,DepartmentofPharmacology,andSchoolofMedicineand§Departmentof
Ophthalmology,ShileyEyeCenter,andInstituteforGenomicMedicine,UniversityofCalifornia,SanDiego,LaJolla,California
92093,¶MolecularMedicineResearchCenterandDepartmentofOphthalmology,WestChinaHospital,SichuanUniversity,
Chengdu610041,China,?DepartmentofOphthalmology,XijingHospital,FourthMilitaryMedicalUniversity,Xi’an710032,China,
**TongRenEyeHospital,Beijing100730,China,‡‡JulesSteinEyeInstituteandDepartmentsofOphthalmologyandNeurobiology,
UCLASchoolofMedicine,LosAngeles,California90095,and§§DepartmentsofBiochemistryandMolecularBiologyand
OphthalmologyandVisualSciences,CentreforMacularResearch,UniversityofBritishColumbia,Vancouver,
British Columbia V6T 1Z3, Canada
Background: Phospholipids containing very long chain polyunsaturated fatty acids (VLC-PUFAs) are enriched in retina.
Results: Specific ELOVL4 rod or cone photoreceptor conditional knock-outs cause decreases in retinal VLC-PUFAs.
Conclusion: ELOVL4 is critical for the synthesis of phosphatidylcholine-containing sn-1 VLC-PUFAs and vision.
Significance: ELOVL4 mutations are implicated in Stargardt disease, a type of juvenile macular degeneration.
Verylongchainpolyunsaturatedfattyacid(VLC-PUFA)-con-
taining glycerophospholipids are highly enriched in the retina;
however,detailsregardingthespecificsynthesisandfunctionof
these highly unusual retinal glycerophospholipids are lacking.
Elongation of very long chain fatty acids-4 (ELOVL4) has been
identifiedasafattyacidelongaseproteininvolvedinthesynthe-
sis of VLC-PUFAs. Mutations in ELOVL4 have also been impli-
cated in an autosomal dominant form of Stargardt disease
(STGD3),atypeofjuvenilemaculardegeneration.Wehavegen-
erated photoreceptor-specific conditional knock-out mice and
usedhighperformanceliquidchromatography-massspectrometry
(HPLC-MS) to examine and analyze the fatty acid composition of
retinal membrane glycerophosphatidylcholine and glycerophos-
phatidylethanolamine species. We also used immunofluorescent
staining and histology coupled with electrophysiological data to
assess retinal morphology and visual response. The conditional
knock-out mice showed a significant decrease in retinal glycero-
phospholipids containing VLC-PUFAs, specifically contained in
the sn-1 position of glycerophosphatidylcholine, implicating the
role of Elovl4 in their synthesis. Conditional knock-out mice were
also found to have abnormal accumulation of lipid droplets and
lipofuscin-like granules while demonstrating photoreceptor-spe-
cificabnormalitiesinvisualresponse,indicatingthecriticalroleof
Elovl4 for proper rod or cone photoreceptor function. Altogether,
this study demonstrates the essential role of ELOVL4 in VLC-
PUFAsynthesisandretinalfunction.
Stargardtdisease(STGD5;fundusflavimaculatus)isthemost
common hereditary form of macular dystrophy and accounts
for 7% of all retinal dystrophies (estimated incidence, 1:10,000)
(1, 2) with patients presenting decreased central vision often in
the first or second decade of life. Ophthalmoscopically, STGD
is characterized by bilateral atrophic changes in the macula,
degeneration of underlying retinal pigment epithelium (RPE),
and the presence of prominent yellowish flecks in the posterior
pole. STGD can be inherited as either a recessive or dominant
trait.Adominantlocushasbeenmappedtochromosome6q14
(STGD3; MIM 600110), and elongation of very long chain fatty
acids-4 (ELOVL4) (GenBankTMaccession number AF277094;
MIM 605512) was identified as the causative gene (3, 4).
The ELOVL4 gene encodes a membrane protein consisting
of 314 amino acids that is predominantly expressed in rod and
cone photoreceptors in the retina and to a lesser extent in the
brain(4).TheELOVL4proteinischaracterizedbyfiveputative
transmembrane segments, a single dioxy iron binding motif
(HXXHH),whichisessentialforenzymaticactivity(5,6),anda
* Thisworkwassupported,inwholeorinpart,byNationalInstitutesofHealth
Grants EY014428, EY018660, and EY019270 (to K. Z.) and EY07042 (to
D. S. W.) from the NEI and Grant U54 GM069338, a LIPID MAPS large scale
collaborative “glue” grant from the NIGMS(to E.A.D.).
1Both authors contributed equally to this work.
2Supported by Research to Prevent Blindness Jules and Doris Stein
Professorship.
3To whom correspondence may be addressed: Depts. of Chemistry and Bio-
chemistryandPharmacology,UniversityofCalifornia,SanDiego,9500Gilman
Dr.,LaJolla,CA92093-0601.Tel.:858-534-3055;E-mail:edennis@ucsd.edu.
4Supported by the National Basic Research Program of China (973 Program,
Grant 2011CB510200), a Veterans Affairs merit award, Research to Prevent
Blindness,aBurroughsWellcomeFundclinicalscientistawardintranslational
research,andtheKingAbdulazizCityforScienceandTechnology(KACST).To
whom correspondence may be addressed: Inst. for Genomic Medicine, Uni-
versityofCalifornia,SanDiego,9500GilmanDr.,LaJolla,CA92093-0838.Tel.:
858-246-0823;Fax:858-246-0961;E-mail:kang.zhang@gmail.com.
5The abbreviations used are: STGD, Stargardt disease; RPE, retinal pigment
epithelium; ELOVL, elongation of very long chain fatty acids; VLC-PUFA,
verylongchainpolyunsaturatedfattyacid;ER,endoplasmicreticulum;PC,
glycerophosphatidylcholine; PE, glycerophosphatidylethanolamine; GIA
sPLA2, Group IA secreted phospholipase A2; ERG, electroretinogram;
ELOVL4,humangene;Elovl4,mousegene;ELOVL4,protein;MRM,multiple
reaction monitoring; FRT, flippase recognition target; DHA, docosa-
hexaenoic acid.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 14, pp. 11469–11480, March 30, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
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carboxyl-terminal dilysine motif (KXKXX) necessary for endo-
plasmicreticulum(ER)retention(7–9).Threealleleshavebeen
identified as the cause for STGD3, each possessing a mutation
inthelastexon(exon6)ofELOVL4.Thesemutationsincludea
5-bp deletion (790–794delAACTT), two 1-bp deletions
(789delT and 794delT), and a single transversion (C-to-G at
position 810) resulting in a premature stop codon (Y270X) (4,
10, 11). Each ELOVL4 mutation results in a truncated protein
withacarboxyl-terminaldilysinemotifabsent,leadingtolossof
retention in the ER; very long chain polyunsaturated fatty acid
(VLC-PUFA) biosynthesis takes place in the ER (12). By defini-
tion, a VLC-PUFA has 24 or more carbon atoms and from four
to six methylene-interrupted cis double bonds (18, 31). When
co-expressed in cell transfection studies, mutant ELOVL4 was
found to exert a dominant negative effect on the wild type pro-
tein, resulting in the mislocalization of the wild type protein to
non-ER juxtanuclear aggregates (13–15).
BasedonthehomologyofELOVL4totheELOfamilyofyeast
proteins as well as other members of the mammalian ELOVL
family and the strong conservation of ELOVL4 between verte-
bratespecies,ELOVL4waspredictedtoplayanessentialrolein
thebiosynthesisofVLC-PUFAs(4,16–21).Cameronetal.(22)
examined the specific elongase activity of ELOVL4 using
homozygous Elovl4 Y270X knock-in and homozygous Elovl4
knock-out mice and observed that mice lacking functional
ELOVL4 died perinatally due in part to dehydration from a
faulty skin permeability barrier. The skin of both the homozy-
gousmutantandtheknock-outmousewasdeficientinglycero-
phospholipidscontainingfattyacids26carbonatomsorlonger
in chain length, implicating ELOVL4 in their biosynthesis.
Agbaga et al. (23) confirmed the metabolic function of
ELOVL4, demonstrating that it is an elongase protein involved
in the synthesis of VLC-PUFAs, particularly those of the poly-
unsaturatedC28–C38n-3variety(Fig.1,AandB).Thiselongase
activitywasstudiedusinganinvitrogain-of-functionapproach
by expressing the mouse ELOVL4 protein in rat cardiomyo-
cytes and human RPE (23). Although ELOVL4 appears to be
expressed only in tissues in which VLC-PUFAs are observed
(24) and it has been noted that the retina is particularly
enriched in VLC-PUFAs (23, 25, 26), no study to date has con-
firmed that ELOVL4 is indeed responsible for the biosynthesis
of VLC-PUFAs in the retina. However, it has recently been
reported that the epidermal expression of an Elovl4 transgene
rescues neonatal lethality of homozygous Stargardt disease-3
mice (27). In the present work, we have generated separately
retinal rod- and cone-specific homozygous Elovl4 knock-out
mice and investigated membrane glycerophospholipid fatty
acid composition, retinal morphology, and physiology. Fur-
thermore, we examined the VLC-PUFA composition in retinal
membrane-intactglycerophospholipidsanddemonstratedthat
the deficiency is specific to glycerophosphocholine (PC) spe-
cies. Our results indicate that ELOVL4 is essential for VLC-
PUFA synthesis as well as retinal functions.
EXPERIMENTAL PROCEDURES
Generation of Photoreceptor-specific Conditional Knock-out
(cKO)Mice—TheDNAfragmentthatcontainedexon2,exon3,
and the flanking region of Elovl4 was cloned from a 129s7/
AB2.2 bacterial artificial chromosome duplicate by homolo-
gous recombination (28). The FRT-flanked neomycin cassette
and the two LoxP sites were inserted into intron 1 and intron 2
through subcloning, whereas the two homologous genomic
fragments, which flanked the cassette and thus mediated the
homologous recombination, replaced exons 2 and 3 in the
endogenous Elovl4 gene. A herpes simplex virus thymidine
kinase gene outside of the 3? homologous arm and from the
backbone of the vector was used as a negative selection marker
in subsequent screenings (Fig. 2A). The final gene-targeting
construct was then electroporated into mouse embryonic stem
(ES) cells derived from 129/SvEv embryonic stem cells (AB2.2,
Stratagene) and cultured in the presence of G418 (positive
selection) or ganciclovir (negative selection). Genomic DNA
was extracted from transfected ES clones to identify homolo-
gous recombination in which the targeted endogenous exons 2
and 3 were replaced with the FRT-Neo-loxp cassette and the
negative selection marker (thymidine kinase) was lost using a
polymerasechainreaction(PCR)andSouthernblotting.Apos-
itiveclone(clone7;Fig.2B)wasinjectedintomouseblastocysts
derived from a C57B/6 background to generate chimeric mice.
The chimeric mice were then bred with wild type C57B/6 (out-
bred) mice to produce heterozygous specimens that were then
genotyped by PCR, incorporating an Elovl4-loxp2-gt-L/Elovl4-
loxp2-gt-R primer. Heterozygous agouti mice were cross-bred
to generate floxed homozygotes. The homozygous Elovl4 lines
having floxed alleles were cross-bred with specimens having
photoreceptor-specific Cre-expressing lines to achieve photore-
ceptor-specific gene inactivation. The cone- or rod-specific Cre
mice(29,30)wereprovidedbyDr.YunLe.Allanimalexperiments
followed the guidelines of the Association for Research in Vision
and Ophthalmology Statement for the use of animals in ophthal-
mic and vision research and were approved by the Animal Care
CommitteeoftheUniversityofCalifornia,SanDiego.
Extraction of Retinal Glycerophospholipids—The removed
retinal tissue samples were placed in a 10-ml borosilicate glass
tubetowhich2mlofice-colddichloromethane/methanol(2:1,
v/v) was added, and then a probe sonicator was used to pulver-
ize the tissue. The tube was then capped and vigorously shaken
for 1 h at room temperature. Two milliliters of water was then
added,andthecontentswerevortexedatmaximumspeedfor1
min.Thetubeandcontentswerethencentrifuged(5000?gfor
2min),andthelower,separatedphasewasremovedandplaced
in another glass tube. This lower liquid phase was stored at
?80 °C until further analysis at which time its contents were
evaporated to dryness with a stream of argon gas, and the
remainingcontentswasresuspendedin100?lofhighperform-
ance liquid chromatography (HPLC) buffer solution A.
Normal Phase Liquid Chromatography of Retinal Extracts—
HPLC was carried out using two Shimadzu (Columbia, MD)
LC-10AD high performance chromatography pumps inter-
faced with a Shimadzu SCL-10A controller. The extracted ret-
inal samples were injected onto the liquid chromatography
analytical column using a Leap Technologies (Carrboro, NC)
PALautosampler.Normalphaseseparationwasachievedusing
a 2.0 ? 250-mm Phenomenex (Torrance, CA) silica column
(Phenomenex catalogue number 00G-4274-B0) held at 40 °C.
Buffer solution A consisted of isopropanol, hexane, 100 mM
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ammoniumacetate(aqueous)(55:40:5,v/v/v);buffersolutionB
consisted of isopropanol, hexane, 100 mM ammonium acetate
(aqueous) (50:40:10, v/v/v). Gradient chromatographic elution
was achieved using 100:0 A/B at 0 min and linearly ramped to
25:75 A/B by 25 min, ramped to 0:100 A/B by 26 min, and held
thereuntil35min.A/Bwasrampedbackto100:0by37minand
held there until 55 min for column equilibration. The buffer
flowratewas0.3ml/min.Separationoptimizationwasachieved
usingPCandglycerophosphoethanolamine(PE)standards.For
each sample analyzed, 40 ?l of sample was injected onto the
column. The HPLC effluent was coupled to a mass spectrome-
ter for further analysis.
FIGURE 1. Analysis of extracted phospholipids from retinas. A, anatomy of a typical very long chain polyunsaturated PC lipid molecule found in retinal
extracts.AVLC-PUFAislocatedonthesn-1positionofthemolecule,whereasDHAoccupiesitssn-2position.ThesePCspeciescontainingVLC-PUFAareunique
in that the proximal carboxylic region (red box) is composed of some 14? saturated carbon bonds, whereas the distal region (blue box) contains up to six
methylene-interrupted cis double bonds. Such species have the length to occupy both halves of a lipid bilayer and would provide quite different physical
propertiestoeachside.B,acombinationofvariousdesaturaseandelongaseenzymesisinvolvedinthesynthesisofVLC-PUFAsbasedontherevisedpathway
ofSprecheretal.(44).TheVLC-PUFAseriescontainingsixdoublebondsisshown;aserieswithfivedoublebondsissimilarlyformed.C,distributionofPCversus
PEspeciesobservedinWTmouseretinalextracts.NotethatthePCspeciescontainingVLC-PUFA(thosehavingansn-1fattyacid32–36carbonatomsinlength)
accountforover10%ofthePCspeciesmonitored;thisisshowningreaterdetailinD.Bycomparison,thePEspeciesobservedinretinalextractscontainhardly
any of these VLC-PUFA species. Data shown in C and D are representative of six independent analyses (each retina from three mice).
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All PC and PE lipid standards used for HPLC and mass spec-
trometry(MS)optimizationwerepurchasedfromAvantiPolar
Lipids (Alabaster, AL). Solvents used for HPLC were of chro-
matography grade and purchased from OmniSolv (Gibbstown,
NJ). Ammonium acetate used as an HPLC additive was pur-
chased from Sigma-Aldrich.
Mass Spectrometry—All mass spectrometric analyses were
performed using an AB Sciex (Foster City, CA) 4000 QTrap
hybrid quadrupole linear ion trap mass spectrometer equipped
with a Turbo V ion source. Acetate anion adducts of the PC
speciesanddeprotonatedanionsofthePEspecieswereformed
by operating the ion source in negative electrospray ionization
mode using the following settings: curtain gas, 10 p.s.i.; nebu-
lizer gas pressure, 20 p.s.i.; heater gas pressure, 20 p.s.i.; ion
spray voltage, ?4500 V; temperature, 500 °C. The mass spec-
trometerwasoperatedinmultiplereactionmonitoring(MRM)
modeusingthefollowingsettings:declusteringpotential,?120
V; entrance potential, ?15 V; collision cell exit potential, ?15
V; collision energy, ?55 V. An MRM method was created and
used that included precursor ion mass/acyl chain fragment
pairs for PC and PE species monitored. AB Sciex mass spec-
trometer software (Analyst 1.5.1) was used to analyze the col-
lected sample data.
In Vitro Group IA Secreted Phospholipase A2(GIA sPLA2)
AssayofRetinalGlycerophospholipids—TheinvitroGIAsPLA2
assay used in this work was modified from assays described
previously (31, 32). Briefly, extracted retinal glycerophospho-
lipids from wild type (WT) mice (as described above) were
dried under argon immediately prior to preparing a mixed
micelle buffer solution for the sPLA2assay. The mixed micelle
buffer was composed of 400 ?M Triton X-100, 50 mM Tris, pH
8.0, and 5 mM CaCl2. The dried retinal lipids were dissolved in
500 ?l of the mixed micelle buffer, incubated at 40 °C for 2 h,
andvortexedevery20minforaperiodof30s.Thismixturewas
theninitiatedbyadding2?lof(0.8mg/ml)GIAsPLA2intothe
mixed micelle substrate and incubated at 40 °C for an addi-
tional 2 h. A control was prepared that was identical to the
above sample except that no sPLA2was added. The glycero-
phospholipids from the two samples were then extracted and
analyzed using HPLC-MS as described above. The GIA sPLA2
was purified from cobra venom (Naja naja naja) as described
previously (33).
Immunofluorescent Staining and Histology—All eye samples
were dissected from adult mice, fixed with 4% paraformalde-
hyde for 2 h at 4 °C, and cryoprotected in a 30% sucrose solu-
tion. Samples were frozen and sectioned (16 ?m) on a cryostat.
For immunohistochemistry, the sections were incubated with
blockingsolution(3%BSAand0.3%TritonX-100inPBS)for30
min followed by overnight incubation with primary antibodies
at 4 °C. Primary antibodies used were Rhodopsin (1:200; Milli-
pore),S-Cone(1:200;SantaCruzBiotechnology),andROM1(a
gift from Dr. R. Molday of University of British Columbia).
AfterrinseswithPBS,sectionswereincubatedwithAlexaFluor
488-conjugated anti-mouse antibody (1:1000; Millipore) and
Alexa Fluor 555-conjugated anti-goat antibody (1:1000; Milli-
pore) for 30 min at room temperature. Sections were counter-
stainedwithDAPIandmounted,andtheimageswerecaptured
using an Olympus FV1000 confocal microscope.
Electron Microscopy—Eyes from 15-month-old wild type
control mice and 15-month-old mutant mice were enucleated,
and their anterior segments were quickly dissected away while
immersed in primary fixative (2% glutaraldehyde ? 2% formal-
dehyde in 0.1 M cacodylate buffer, pH 7.4). The resulting eye-
cups were maintained in primary fixative for ?24 h, then post-
fixedin1%OsO4inthesamebuffer,dehydrated,andembedded
inEpon.Semithinsectionswerestainedwithtoluidineblueand
imaged by light microscopy. Ultrathin sections were stained
withuranylacetateandleadcitrateandimagedbytransmission
electron microscopy.
Electroretinography—Electroretinograms (ERGs) were ob-
tained from adult mice in a full-field dome using methods sim-
ilar to those in clinical practice and using stimuli comparable
with standards cited by the International Society for Clinical
ElectrophysiologyofVision.Themiceweredark-adaptedover-
night and prepared for recording the next day under dim red
light conditions. ERG recordings were conducted as described
previously (13). In brief, anesthesia was induced using an intra-
peritoneal injection of a mixture of ketamine (90 mg/kg) and
xylazine(9mg/kg)insalinesolution.Pupilsweredilatedusinga
mixture of tropicamide (1%) and phenylephrine (2.5%). Propa-
racaine (0.5%) was used as a topical anesthetic to minimize
blinking. Mice were then placed on a heating pad in a Ganzfeld
bowl stimulator on a sliding table. ERGs were recorded using a
loopedthinstainlesssteelwiretomakecontactwiththecorneal
surfacethroughathinlayerofGentealTearGel(NovartisPhar-
maceuticals). Needle electrodes placed in the lower lip and tail
served as reference and ground leads, respectively. Amplifica-
tion with a Grass 15LT external amplifier (at 0.3–300 Hz with-
out notch filtering), stimuli presentation, and data acquisition
were programmed and performed using the VERIS 6.0.9 Sci-
ence software system (Electro-Diagnostic Imaging, Inc.,
Redwood City, CA). Single flashes (0.05-ms duration (0.0084
Candela?s total) with 10-s intervals for 10 counts in scotopic
testing, 1.0-ms duration (2.686 Candela?s per square meter
total) with 10-s intervals for 10 counts in mixed testing, and
1.0-msduration(2.686Candela?spersquaremetertotal)with
100-ms intervals in photopic flicker testing) were obtained
under both dark-adapted conditions and light-adapted condi-
tions.ForphotopicERGresponses,micewerelight-adaptedfor
aminimumof10minpriortorecording.Micewereplacedona
heating pad and visually observed until the effects of the anes-
thetics had diminished entirely.
RESULTS
Generation of Rod- or Cone-specific Elovl4 Knock-out Mice—
After the Elovl4 gene-targeting construct was electroporated
into mouse ES cells and cultured in the presence of G418 (pos-
itive selection) and ganciclovir (negative selection), genomic
DNA was extracted for Southern blotting analysis with the
external probe (Fig. 2A, purple bar). Of eight transfected ES
clones, our result demonstrated the presence of the mutant
constructasindicatedbythe4.7-kbband(Fig.2B,lane7).After
cross-breeding FRT-Neo-loxP mice with ACTB-FLP mice to
remove the neomycin marker, genomic DNA was obtained
frommouselinesforPCRgenotypingtoconfirmthecreationof
a conditional floxed Elovl4 allele (Fig. 2C). Homozygous Elovl4
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lines with floxed alleles were then cross-bred with mice having
photoreceptor-specificCre-expressedlinestoachievethepho-
toreceptor-specific Elovl4 gene knock-out specimen.
Analysis of Retinal Extract Glycerophospholipids—Extracts
obtained from WT mouse retina were analyzed for a total of 21
different PC and PE molecular species (42 total), including
those containing both five- and six-double bond VLC-PUFA
acylchains;theseresultsareshowninFig.1,CandD.Aninitial
surveyrevealednosignificantglycerophospholipidspeciescon-
taining VLC-PUFA with fewer than five or more than six dou-
ble bonds present (data not shown), so these species were not
monitored in subsequent retinal surveys. An HPLC-MS MRM
method was created that included precursor anion mass/acyl
chain fragment anion mass MRM pairs specific for these 21 PC
andPEspecies.TherelativepercentabundanceshowninFig.1,
C and D, was determined by integrating the area under each
MRM defined peak, separately summing for the PCs and PEs,
and calculating the percentage of each molecular species com-
prising the PC and PE totals; this assumes that ionization and
fragmentation efficiencies for the different molecular species
are similar. Comparing the PCs with the PEs, in addition to
showing a difference in the distribution of the various species,
these results also show that the vast majority of the glycero-
phospholipidscontainingVLC-PUFAsarerestrictedtothePCs
with those containing C32–C36VLC-PUFAs comprising over
10% of the 21 species monitored. These results are the average
of six independent analyses (each retina from three mice) and
are in agreement with earlier studies (34, 35).
Anabsolutequantitative
amount/mg of retinal tissue) of the PC and PE species moni-
tored was not feasible as no deuterium-labeled internal stand-
ardsarecurrentlycommerciallyavailableforanyoftheglycero-
phospholipids containing VLC-PUFA acyl chains, and it is for
this reason that we report relative abundances in our current
work. However, using accurately measured primary standards
forsomeofthemoreabundantshorterchainPCandPEspecies
(e.g. 16:0/18:1), we estimated the total PC abundance to be
?5-fold higher compared with the total PE abundance.6
To confirm an earlier study (34) showing that VLC-PUFA
acylspecieswerecontainedalmostexclusivelyonthesn-1posi-
tion of retinal PCs with docosahexaenoic acid (DHA; 22:6n-3)
measurement(e.g.pmol
6Using commercially available 16:0/18:1-PC and 16:0/18:1-PE standards,
eachwithaccuratelyknownconcentrations,weobservedthatequalabso-
lute quantities of each standard produced approximately the same inten-
sity mass spectral monitoring signal peak (MRM pair: precursor anion
mass/acylchainfragmentanionmass).Thus,forourretinalsamples,signal
intensities obtained from PC and PE species having the same two acyl
chain moieties could be used to estimate an absolute -fold difference,
allowingustoestimatethetotalPCabundancetobeapproximately5-fold
higher compared with the total PE abundance.
FIGURE 2. Elovl4 cKO construct. A, the schematic diagram of the Elovl4 locus and targeting construct. An FRT-flanked neomycin (Neo) cassette positive
selectionmarkerwasinsertedintointron1.Exons2and3wereflankedbytwoLoxPsites.Thereisaherpessimplexvirusthymidinekinase(TK)counterselection
cassetteinthe5?-endofthevector.ThewildtypebandexpectedfromBamHIdigestionis12.8kb;themutant(MT)bandis4.7kb.B,Southernblotanalysisof
ES cell genomic DNA after digestion with BamHI and hybridization with 3? external probes. Left of the molecular weight marker is genomic DNA from
untransfectedEScells,andlanes1-8aregenomicDNAfromclones1–8withtransfectedtargetingconstruct.C,PCRgenotypingofagoutimousegenomicDNA.
A single 271-bp fragment appears in the case of wild type mice, and an additional 355-bp fragment appears in the case of heterozygous mutant mice.
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occupying the sn-2 position, an in vitro GIA sPLA2assay was
carriedoutinthecurrentwork,andtheresultsareshowninFig.
3. An HPLC-MS MRM method was created that included pre-
cursor acetate adduct anion mass/acyl chain fragment anion
mass MRM pairs as well as their corresponding lyso analogs
(result of cleaving the VLC-PUFA or the DHA acyl chain from
these PCs). The same HPLC-MS-MRM method was used to
monitor both the control (Fig. 3A) and the sPLA2-treated (Fig.
3B)samples.Asexpected,PCspeciescontainingboththeintact
VLC-PUFAandtheDHAacylchainswereobservedinthecon-
trol sample, and these were not observed in the sPLA2-treated
sample. The sample treated with the sPLA2showed the pres-
enceofVLC-PUFA-containinglyso-PCspecies.BecausesPLA2
is specific for cleaving the sn-2 acyl chain (36), these results
confirm that the VLC-PUFAs are indeed located on the sn-1
position of PC. Lyso-PC-containing DHA was also monitored;
however, its minimal increase in the sPLA2-treated sample
would imply that it was not located on the sn-2 position of the
VLC-PUFA-containing PCs.
Next, the retinal extracts from both mouse Rod-cKO and
Cone-cKO samples were assayed for the 21 PC species and
comparedwithmouseWTsamples.Therelativepercentagesof
PCspecies(Fig.4,AandB)weredetermined,anddifferencesin
each PC molecular species among Rod-cKO versus WT and
Cone-cKOversusWTareshownasaratiodifferenceinFig.4C.
Thosespeciesfallingonorneartheheavydashedhorizontalbar
at a ratio equal to 1 are approximately the same among the
cKOs versus WT (e.g. 16:0/18:1 and 18:0/22:6), those above the
bar are higher in the cKOs versus WT (e.g. 22:6/22:6 and 22:6/
22:5),andthosebelowthebararelowerinthecKOsversusWT
(e.g. 22:6/34:6 and 22:6/36:6). Fig. 4D shows the proposed syn-
thetic pathway shown earlier in Fig. 1B; highlighted in blue are
those species higher in the Rod-cKO compared with WT,
whereas highlighted in red are those species lower in the Rod-
cKO compared with WT.
Retinal Morphology in Photoreceptor-specific Elovl4 Condi-
tional Knock-out Mice—Immunohistochemistry and confocal
microscopy carried out on the frozen sections of the retinas of
3–5-montholdWT,Rod-cKO,andCone-cKOmiceshowsim-
ilar retinal morphology in both photoreceptor-specific cKOs
compared with WT (Fig. 5, A–F). Gross morphology was nor-
mal (Fig. 6, A and B), although there was a mild loss of photo-
receptorcellsintheRod-cKOretinas.Countsofnuclei(500?m
either side of the optic nerve head in dorsoventral sections)
revealed that 10-month-old Rod-cKO mutants had eight rows
ofnuclei,and15-month-oldRod-cKOmutantshadsevenrows
ofnucleiincomparisonwithninerowsofnucleifor15-month-
old normal controls. The disk membranes of the rod outer seg-
FIGURE3.InvitroGIAsPLA2assayofretinalglycerophospholipids.TheHPLC-MSMRMchromatogramresultsforextractedmouseWTretinalglycerophos-
pholipid control (A) and GIA sPLA2-treated sample (B) show that the VLC-PUFAs are located exclusively on the sn-1 position of retinal PCs. The chemical
structuresforC32–C36VLC-PUFA-containingPCsareshownalongwiththeiracetateadductanionprecursormassesandthesn-1andsn-2acylchainfragment
masses(MRMpairs).Theircorrespondinglyso-PCanalogs,theresultofsPLA2cleavingthesn-2-locatedDHA,arealsoshown.Rel.,relative.Errorbarsrepresent
S.E.
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ments in the Rod-cKO mutants (Fig. 6C) and the cone outer
segments in the Cone-cKO mutants appeared normal for
immersion-fixed mouse retinas. In the RPE of both Rod- and
Cone-cKO retinas, an accumulation of lipid droplets was evi-
dent (Fig. 6, E–G); such droplets were only rarely observed in
the RPE of normal control retinas (Fig. 6D). Lipofuscin-like
granules were also observed in the RPE of the Cone- and espe-
cially the Rod-cKO retinas (Fig. 6, E and F); such granules were
also evident in 15-month-old control retinas, although in the
controls, they were clearly much less abundant.
Deterioration of Electrophysiological Responses in Condi-
tional Knock-out Elovl4 Mice—ERG recordings were per-
formed to assess retinal function in Elovl4 Cone-cKO, Elovl4
Rod-cKO, and WT adult mice (Fig. 5, G–I). For the Rod-cKO
mice, we found a significant reduction in the maximum rod
b-wave response (163 ? 19 microvolts (?V)) relative to WT
(238?18?V)(p?0.011)andasignificantreductioninrodand
cone mixed b-wave response (243 ? 16 ?V) when compared
withWT(316?22?V)(p?0.017).Cone-cKOmiceshoweda
significant reduction in flicker response (24 ? 4 ?V) when
FIGURE4.DifferentialanalysisofeachPCspeciesinretinaofRod-andCone-cKOmice.A,relativeabundanceofPCspeciesobservedinWT,Rod-cKO,and
Cone-cKO mouse retinal extracts. B, greater detail of the region representing the PC species containing 32–36 VLC-PUFAs. C, graph showing the comparison
ofPCspeciesobservedintheretinalextractsofRod-andCone-cKOcomparedwithWTmice(Rod-cKOisshowninred;Cone-cKOisshowningreen).Notethe
heavydashedhorizontalbarindicatingaratioof1.PCspecieslocatedabovethislineindicatehigherlevelsobservedincKOcomparedwithWT;thoselocated
below indicate lower levels observed in cKO compared with WT. D, the VLC-PUFA synthesis pathway showing the species that were found to be higher in
Rod-cKO mice compared with WT highlighted in blue and those that were found to be lower highlighted in red. Data shown are representative of six
independent analyses (each retina from three mice). Error bars represent S.E.
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compared with WT (43 ? 4 ?V) (p ? 0.004). The photorecep-
tor-specific pattern of reduction in the visual response
recorded for cKO mice indicates that Elovl4 is essential for the
rod or cone photoreceptors to function properly in the retina.
Additionally, the Rod-cKO mice demonstrate significant
reductions in rod function, and the Cone-cKO mice demon-
strate significant reductions in cone function.
DISCUSSION
Using photoreceptor-specific conditional knock-out mice,
we provide evidence to implicate Elovl4 as being essential for
VLC-PUFA synthesis. In the current work, HPLC-MS analysis
of retinal extracts from WT mice supports earlier studies (34),
confirming that VLC-PUFA-containing glycerophospholipid
species(C32–C36)arerestrictedalmostentirelytothePCswith
these VLC-PUFA PCs accounting for roughly 10% of the 21 PC
species we monitored (Fig. 1, C and D). We also observed dis-
tribution profile differences when comparing these 21 moni-
tored PC and PE species (Fig. 1C) and estimated the PC species
tobe?5-foldhigherinabsoluteabundancecomparedwiththe
PE species.
The occurrence of VLC-PUFA acyl species contained in ret-
inal glycerophospholipids was first observed over 20 years ago
by Aveldaño (34). In agreement with the current work, this
earlier study showed that the occurrence of VLC-PUFAs is
restricted almost entirely to PC species found in the retina.
Using a phospholipase A2enzyme, we also confirmed Avelda-
ño’s (34) earlier work showing these VLC-PUFA acyl species
were contained almost exclusively on the sn-1 position of these
PCs with DHA (22:6n-3) occupying the sn-2 position. In the
current study, we observed these VLC-PUFAs to be highly
polyunsaturated, having mostly six double bonds, to a lesser
extent five double bonds, and essentially no acyl chains with
greaterthansixorfewerthanfivedoublebonds.Studies(22,23,
26,35)subsequenttothatofAveldaño(34)investigatingretinal
glycerophospholipids have for the most part used gas chroma-
tography-mass spectrometry (GC-MS) methodology for analy-
sis.NotethatGC-MSanalysisofglycerophospholipidsrequires
theacylfattyacidstobefirstdeacylatedfromtheircorrespond-
ing glycerophospholipid precursor molecule, thus creating
uncertaintyastotheirexactprecursormoleculeoforigin.Once
these fatty acids are deacylated and in “free form,” they are
FIGURE5.NormalgrossmorphologyofretinainRod-orCone-cKOmicewithdeteriorationinphotoreceptor-specificelectrophysiologicalresponse.
Retinas from 3–5-month-old adult wild type (A and D), Rod-cKO (B and E), or Cone-cKO (C and F) mice were double fluorescence immunostained with
Rhodopsin (green) and S-Cone (red; A–C) or ROM1 (green) and S-Cone (red; D–F). Nuclei were counterstained with DAPI. Inset in E shows a magnified image of
the boxed area. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; IS, inner segment of photoreceptors; OS, outer segment of photore-
ceptors. G–I, ERG responses from C57B/6 WT control, Elovl4 Rod-cKO, and Elovl4 Cone-cKO mice. G, average maximum scotopic b-wave amplitudes to stimuli
thatelicitresponsesonlyfromrods.H,averagemaximummixedb-waveamplitudestostimulielicitingrodandconeresponses.I,averagemaximumconewave
amplitudesin10-HzphotopicflickerERG.Verticalaxisunitsarein?V.N.S.,notsignificant.ErrorbarsrepresentS.E.,n?11forwildtype,n?18forRod-cKO,and
n ? 10 for Cone-cKO.
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pooledforanalysis,makingitimpossibletoidentifythespecific
glycerophospholipid from which they originated. A more
recent study investigating retinal glycerophospholipids used
HPLC directly coupled to the mass spectrometer operating in
positive electrospray ionization mode, which allowed for the
analysis of intact glycerophospholipids (37). In that study, reti-
nal PCs were analyzed as protonated cations and collisionally
induced dissociation fragmentation in the mass spectrometer
of these cations yields only one detectable fragment ion: the
phosphocholine headgroup with a mass-to-charge (m/z) ratio
of 184. Additionally, the fragmentation of a protonated PE cat-
ionyieldsonlyonedetectablefragmentionwithamassequalto
the precursor ion less the mass of the PE headgroup (less than
141).Thus,theinformationprovidedindicatesonlytheprecur-
sor ion mass and the specific glycerophospholipid class (PC or
PE)towhichitbelongs,leavinguncertaintyastotheexactiden-
tity of the specific acyl chains of the molecule.
An alternative mass spectrometric approach and the one
used in the current work uses negative electrospray ionization
toformanions(acetateanionadductsofPCsanddeprotonated
anions of PEs), which upon collisionally induced dissociation
fragmentation produce intact acyl chain anion fragments,
FIGURE6.MicroscopyshowingnormalretinalmorphologybutabnormalaccumulationsoflipofuscinandlipiddropletsinRPE.Retinasfrom15-month-
oldRod-orCone-cKOandwildtypemicewerestudied.AandB,lightmicrographsofsemithinsectionsfromacontrolretina(A)andaRod-cKOretina(B).Both
sectionsaretaken0.5mmfromtheopticnervedhead.Scalebars,20?m.C,electronmicrographsofthedistalendsofrodoutersegmentsfromaRod-cKOretina
thatwasfixedbyimmersion;thediskmembranesappearnormalforthisfixationprocedure.D–G,electronmicrographsoftheRPE(apicalsurface,upper;basal
surface,lower)fromacontrolretina(D),aCone-cKOretina(E),andaRod-cKOretina(FandG).Scalebars,1?m(C–F)and500nm(G).Lipiddroplets(arrowheads)
are much more abundant in both the Rod- and Cone-cKO RPE. These retinas also contain somewhat more lipofuscin (arrows).
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allowing for a definitive characterization of the glycerophos-
pholipid molecule (34). It is also noted that although in some
cases the precise sn-1 and sn-2 positions of these glycerophos-
pholipid acyl chains can be inferred by their relative fragment
intensity ratios (38) this is not an exact rule, and numerous
exceptions have been observed (38). We therefore used an
sPLA2assaytoconfirmthattheVLC-PUFAsarelocatedonthe
sn-1 position of the retinal PCs. We also compared the retinal
extracts of WT mice with those deficient in either cone or rod
ELOVL4 (Cone-cKO or Rod-cKO) and observed both the
Cone- and Rod-cKO mice to have increases in some of their
intermediate length (C20–C24) PUFA-containing PCs, which
are presumably precursors of the VLC-PUFAs as shown in Fig.
1B; a concomitant decrease in those PCs containing C28–C36
(Rod-cKO) and C32–C36(cone-cKO) VLC-PUFAs; and closely
similar amounts of the most abundant shorter acyl chain PCs
(e.g. 16:0/18:1 and 18:0/22:6; Fig. 4, A–C). The more dramatic
decreaseintheVLC-PUFAPCsobservedintheRod-cKOcom-
pared with the Cone-cKO (Fig. 4C) is likely due to the 10–20-
fold higher abundance of rod cells compared with cone cells in
the retina, and note that the Cone-cKO mice still maintained
ELOVL4-active rod cells, and the Rod-cKO mice still main-
tained ELOVL4-active cone cells. The higher abundance of the
intermediate PC species, particularly the 20:4/22:6, 22:6/22:6,
and 22:6/22:5 in the cKO mice compared with the WT may be
due to a “backup” effect because these precursors were not fur-
ther elongated to their VLC-PUFA products (Fig. 4D).
Although these intermediate species only account for ?5% of
thespeciesmonitored,itispossiblethattheirhigherabundance
in the cKO mice could have some detrimental effect.
At present, the precise function of VLC-PUFA PCs in the
retinaremainslargelyunknown.Theymayperformastructural
role in the lipid bilayer, such as forming a domain or rafts to
facilitate protein interactions. The exclusive association of the
phosphocholineheadgroupandthesn-1VLC-PUFAacylchain
may confer special properties to these unusual molecules in a
lipid bilayer. For example, Agbaga et al. (23) proposed that the
VLC-PUFA acyl chain may extend and cover the entire bilayer,
thereby providing a flexible hinge at the rim location where the
curvature of photoreceptor disk membranes is greatest. The
abnormal accumulation of lipid droplets and lipofuscin gran-
ules observed in the RPE of mutant retinas may be related to
defective retinoid mobility at the disk rim. Excessive lipofuscin
in the RPE of Abca4?/? mice, a model for STGD1, appears to
result from a lack of N-retinylidene-PE flippase activity of the
ABCA4 protein in the disk membrane rims (39). Alternatively,
it is possible that VLC-PUFAs perform a direct signaling func-
tion: Bazan et al. (40) have proposed that lipid molecules, such
as DHA, serve as a special class of signaling molecules that
activatespecificreceptors.Kahn-Kirbyetal.(41)havereported
that eicosapentaenoic acid and arachidonic acid can modulate
transient receptor potential cation channel activity and modu-
late olfactory and nociceptive behavior.
At the light microscopy level, it appeared that the overall
layered structure of the retina is not affected by the cell type-
specificeliminationofElovl4.However,acloserexaminationof
electron micrographs revealed some abnormalities in the RPE,
including an excessive accumulation of lipofuscin and lipid
droplets (Fig. 6). These accumulations may result from
impaired digestion of photoreceptor disk membranes, which
contain an altered balance of fatty acid precursors, due to the
loss of ELOVL4.
Defects in our electrophysiological studies (ERGs) in Elovl4
knock-out mice are consistent with its role in signal transduc-
tion.Thereareexamplesofgeneswhoselossoffunctionaffects
ERGs but not morphology (42, 43). Alternatively, the lack of
fatty acid elongation in ELOVL4 knock-out cells may affect
multiple cellular processes, such as ER function, endocytosis,
andsecretion.TheidentificationofthepreciseroletheseVLC-
PUFAPCsplayshouldproveinformativetoourunderstanding
of retinal normal physiology and degeneration.
Lastly, we raise the question as to what possible mechanisms
couldbeinvolvedinthegenerationofthesemostunusualVLC-
PUFA-containing glycerophospholipids found in the retina.
Based on the revised pathway of Sprecher et al. (44), Fig. 1B
shows that a combination of various desaturase and elongase
enzymes is involved in their biosynthesis. Although plants and
fungi have “lipid-linked” desaturases that are capable of acting
on the intact acyl chains of glycerophospholipids substrates
(45–47), desaturation and elongation of fatty acids in verte-
brate systems requires an acyl-CoA substrate (48, 49) and the
fatty acid to be free of any glycerophospholipid attachment.
Because a number of desaturase and elongase reactions are
required in coordinated and sequential steps to generate a
VLC-PUFA from its initial precursor (e.g. 18:3n-3), it seems
mostlikelythatthesereactionsoccurpriortoanyglycerophos-
pholipidattachment,thuscreatingavariedyethighlyregulated
pooloffattyacidsthatarethenmethodicallyplacedonthesn-1
positionoftheglycerophosphatedestinedtoformtheresulting
PC through the action of specific acyltransferases.
In an alternative scheme, as the precocious, “waiting-to-be”
VLC-PUFA journeys through its sequential desaturation/elon-
gation steps, it is also possible that it can periodically be acy-
lated to a glycerophosphocholine or lysoglycerophosphocho-
line to form a glycerophospholipid and hence is “off-limits” to
desaturation/elongation during this time until once again it is
deacylated and subjected to another round of desaturation/e-
longation. Because it is likely that both the desaturation/elon-
gation steps and the acylation/deacylation steps occur in the
endoplasmic reticulum, this scenario would not require the
intermediate components to be constantly shuttled back and
forth across organelles. However, more steps are involved in
suchaschemecomparedwiththealternativeschemedescribed
in the previous paragraph, so if it does indeed occur, then one
would need to ask what advantages may be gained. It is also
possible that the VLC-PUFAs are biosynthesized through
eitheroneoracombinationofbothschemesandthenincorpo-
rated into “more typical,” shorter acyl chain glycerophospho-
lipid molecules through an acyl chain remodeling process (50,
51).
It is important to understand how VLC-PUFAs function at
the molecular level to affect the functioning of the rods and
cones. Our results show that the presence of some amount of
PC containing a VLC-PUFA at the sn-1 position is essential for
the visual process. Perhaps these special PCs play a biophysical
role by their position in the lipid bilayer membrane, and their
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lack is sufficient to distort its normal membrane structure or
phase, thereby affecting the visual signal transduction process.
Although we could not detect such structural affects in the
microscopic imaging studies used in the current work, future
retinal work involving newly developing imaging mass spec-
trometry(52,53)mayprovideusefulinformationforinvestigat-
ingthespatialandcellulardistributionofglycerophospholipids
containing VLC-PUFAs in individual rod and cone retinal cells
and elucidation of their visual functions in health and disease.
Acknowledgments—We thank Kevin Wang and Bin Lin for technical
assistance,PeterShawandothermembersoftheZhanglaboratoryfor
helpful discussions, and Dr. Yun Le and Gene Anderson for providing
thecone-orrod-specificCremice.E.A.D.thanksProfessorMichaelH.
Goldbaum at the University of California, San Diego Shiley Eye Cen-
ter for special insight and help in understanding the pathophysiology
and clinical implications of retinal disease.
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